Abstract:
:The yeast Pichia pastoris is a widely used host organism for heterologous protein production. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number integrants of P. pastoris are achievable only by screening of random events or by cloning of gene concatemers. Methods for rapid and reliable multicopy integration of the expression cassette are therefore desirable. Here we present such a method based on vector integration into the rDNA locus and post-transformational vector amplification by repeated selection on increased antibiotic concentrations. Data are presented for two exemplary products: human serum albumin, which is secreted into the supernatant, and human superoxide dismutase, which is accumulated in the cytoplasm of the cells. The striking picture evolving is that intracellular protein production is tightly correlated with gene copy number, while use of the secretory pathway introduces a high clonal variability and the correlation with gene copy number is valid only for low gene copy numbers.
journal_name
FEMS Yeast Resjournal_title
FEMS yeast researchauthors
Marx H,Mecklenbräuker A,Gasser B,Sauer M,Mattanovich Ddoi
10.1111/j.1567-1364.2009.00561.xsubject
Has Abstractpub_date
2009-12-01 00:00:00pages
1260-70issue
8eissn
1567-1356issn
1567-1364pii
FYR561journal_volume
9pub_type
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